The members of nuclear factor (NF-kB) transcription factor family play central roles in various biological processes by regulating expression of genes involved in such diverse processes as inflammation, immune responses, differentiation, proliferation, and apoptosis. The primary objective of our research is to characterize the function and regulation of NF-kB, specifically the characterization of the mechanisms of regulation and termination of IKK/NF-kB activation in response to upstream signaling by Tumor Necrosis Factor-a (TNF-a) and Interleukin-1 (IL-1). Towards that end we have followed different experimental approaches.1) We have identified two proteins that not been reported to modulate NF-kB activity. Both these proteins contain phosphoinositol-3-phosphate (PI3P) binding FYVE domain and ubiquitin ligase RING (E3) domain. These proteins CARP-1 and -2 (caspase-8 and -10-associated RING proteins) bind to and negatively regulate DED caspases. These proteins are localized to different compartments in endocytic pathway and negatively regulate the TNF-a induced NF-kB. Our studies also indicated that CARPs downregulate TNF-R1 signaling by directly binding to and significantly downregulating RIP protein expression, a critical component of the TNF-R1 receptosome. siRNA-mediated downregulation of CARPs in two different cell lines enhanced TNF-a induced NF-kB activation. Preliminary results with siRNA -mediated downregulation of CARP-1 in H460 cell line indicated a significant delay in the recovery of I?B level leading to the prolongation of TNF-a induced NF-kB activation. 2) Yeast two-hybrid screens using CARP-1 as bait to identify its partners in NF-kB activation has resulted in the identification of several interacting molecules. Initial results indicate that one of them (an unknown protein) is a plasma membrane protein with seven trans-membrane domains. Over expression of this protein resulted in negative regulation of TNF-a induced NF-kB activation. We have also found a second interactor that acts as a positive regulator of the same signaling pathway. We will continue our focus on the role played by CARPs in TNF-R1 internalization/endocytosis and the regulation or termination of IKK/NF-kB activation.